Detection of malarial parasites in blood

ABSTRACT

PRE-STAINED MICROSCOPE SLIDES HAVING A SURFACE COVERED WITH A DRIED MIXTURE OF METHYLENE BLUE NN (ALSO KNOWN AS NEW METHYLENE BLUE N) AND CRESYL VIOLET ACETATE, DISCLOSED IN A COPENDING APPLICATION AS USEFUL FOR OBSERVING NORMAL BLOOD CONSTITUENTS, ARE EMPLOYED TO OBSERVE SAMPLES OF BLOOD FROM PATIENTS SUFFERING FROM FALCIPARUM MALARIA. TROPHOZOITES AND GAMETOCYTES ARE RENDERED READILY OBSERVABLE AND IDENTIFIABLE; PERSONNEL EXPERIENCED IN MALARIAL-ORIENTED HEMATOLOGY FOUND THE PARASITES IN SUCH PREPARATIONS AT LEAST READILY OBSERVABLE AS IN THE CONVENTIONAL WRIGHT-STAINED PREPARATION.

3,834,874 DETECTION OF MALARIAL PARASITES IN BLOOD Stanley Gottlieb,Philadelphia, and John A. Geating, Abington, Pal, assignors to GeneralElectric Company No Drawing. Filed Oct. 18, 1972, Ser. No. 298,549 Int.Cl. C03c 17/00; G01n 1/30, 33/16 U.S. Cl. 23-230 B 2 Claims ABSTRACT OFTHE DISCLOSURE CROSS-REFERENCE TO RELATED APPLICATION Pre-Stained Slidesfor Blood Tests, John A. Geating and Frederick W. Thomae, Jr., Ser. No.222,654, filed Feb. 1, 1972, now US. Pat. 3,796,594, assigned to theassignee of the present application.

BACKGROUND OF THE INVENTION (1) Field of the Invention This inventionpertains to methods of biological diagnostic observation, and inparticular to a method of identifying the presence of malarial parasitesin human blood.

(2) Prior Art U.S. Pat. 3,796,594 there is described a pre-stainedmicroscope slide which has on one surface a dried mixture of CresylViolete Acetate and Methylene Blue NN in suitable proportions andsurface density so that when a drop of blood is applied to the slide andspread over it by application of a cover glass, the normal components ofthe blood are differentially stained, producing the followingcharacteristics:

Neutrophils contain a finely granulated cytoplasm which stains a lightpurple in the fresh preparation and fades to a greenish tint in theolder smear. Nuclei stain a bright purple.

Eosinophils exhibit a bright purple nucleus and a cytoplasm packed withlarge uniform orange-staining granules which fade to a greenish orangewhen the preparation is about twelve hours old.

Basophils contain smaller number of uniform-size granules thaneosinophils, which stain a dark purple and often completely obscure thenucleus. With focusing, an orange tinge can be observed in the granulesfound at the edge of the cell, an effective identifying feature.

Lymphocytes develop a purple staining nucleus surrounded by a lighterpurple cytoplasm which may become as dark as the nucleus as thepreparation grows old.

Monocytes have an affinity for the stain similar to lymphocytes.Distinguishing features are the larger size and the greater amount ofcytoplasm of the monocytes. It has been found that the differentiationis readily learned.

Platelets stain a pink color. A platelet estimation is best performed assoon after the staining period as possible because of an accumulation ofdebris with increasing time.

Reticulocytes contain a reddish-purple network but do not fade evenafter the red cells become distorted. Never- United States Patent icetheless, the reticulocyte count should also be made within 4 to 5 hoursafter the staining period because of the fading of some of the red bloodcorpuscles when they die.

The published prior art, including that used for observation of bloodfor suspected malarial infection, involves the use of Wrights stain. Apublication entitled The Mor phology of Human Blood Cells in WrightStained Smears of Peripheral Blood and Bone Marrow, by L. W. Diggs,M.D., Dorothy Sturm, and Ann Bell, University of Tennessee College ofMedicine, Department of Medicine, Section of Hematology and City ofMemphis Hospitals, Memphis, Tenn., copyright 1970, published by AbbottLaboratories, North Chicago, 111., at page 35 thereof containsillustrations of various samples stained in the conventional manner withWrights stain.

SUMMARY OF THE INVENTION Samples of blood are applied to the pre-stainedslides described in US. Pat. 3,796,594, to which reference is made fordetails not given herein. In addition to the staining of normal bloodconstituents described therein, trophozoites are stained so that theyare readily observed not only in erythrocytes, but alsoextra-erythrocytically, which latter has not previously been observed,so far as the applicant is aware. It is possible that the presumedfailure to make the latter observation in the prior art reflects afailure of the conventional stains of the prior art to render themsufficiently visible to be detected, although this has not been thesubject of investigation.

DESCRIPTION OF THE PREFERRED EMBODIMENT Samples of blood from threehospitalized patients receiving anti-malarial therapy, and from threepatients suspected of having Falciparum Malaria were made intoconventional thin preparations stained with Wrights stain, and samplesfrom the same six patients were applied to prestained slides having drymixed stains on their surfaces, as described in detail in US. Pat.3,796,594. Briefly, a solution of 2.0 grams Methylene Blue NN in 25milliliters of absolute methyl alcohol, and a solution of 1.0 gramCresyl Violet Acetate in 25 milliliters of absolute methyl alcohol, arefiltered separately after standing from 16 to 24 hours. One milliliterof the first named solution and 0.13 to 0.2 milliliters of the secondsolution are mixed and a clean microscope slide is coated with a film ofthe mixture and allowed to dry in air, leaving a dried film of 91 to 94percent by weight Methylene Blue NN and 6 to 9 percent by weight ofCresyl Violet Acetate. No malarial parasites were observed in either theWright-stained preparation or the pre-stained slide preparation of thesamples from the patients receiving therapy. Parasites observations ofthe blood of one of the suspected patients were also negative for boththe Wright-stained preparation and the pre-stained slides. Samples ofblood from the remaining two suspected patients showed identifiablemalarial parasites both in the conventional Wright-stained preparationsand in the pre-stained slides. Several observers, including a doctor ofmedicine and a research assistant from a university faculty of tropicalmedicine, both with considerable experience in the identification ofmalarial parasites, confirmed all the findings described herein.

The mode of preparation of all samples applied to prestained slides, andthe observations of such preparations in the case of samples containingmalarial parasites was as follows:

A drop of blood was obtained from the finger and placed on thepre-stained slide. A 22 by 40 mm. cover slip was placed over the drop ofblood which resulted in spreading of the drop of blood under the coverslip. After approximately fi've minutes the slides were placed on themicroscope and were examined under both high dry magnification and oilimmersion and the following observations were made:

Comparison was made with the illustrations found on page 35 of TheMorphology of Human Blood Cells in Wright Stained Smears of PeripheralBlood and Bone Marrow, by L. W. Diggs, M.D., Dorothy Strum, and AnnBell, University of Tennessee College of Medicine, Department ofMedicine, Section of Hematology and City of Memphis Hospitals, Memphis,Tenn, copyright 1970, Abbott Laboratories, North Chicago, Ill. Of thetwo cases observed, one individual was found to have a light infectionand the other was found to have a very heavy infection.

Trophozoites, both early and late, were observed in the smears of bothindividuals. The chromatin dot in the ring forms stained purple tored-purple in color as compared to the conventional staining method. Thecytoplasm was slightly pink in color. In the light infection thetrophozoites appeared more spherical and smaller in size as compared tothe standard method in which they appeared larger and more ovoid. In thecase of the heavy infection, many trophozoites appeared ovoid as in aconventional preparation but they did, however, appear smaller.

In several instances ring forms were detected extra-erythrocytically.The experienced physician previously mentioned stated that trophozoitesare not known to occur extra-erythrocytically and that he had neverobserved it previously. Many erythrocytes contained two parasites, acondition which is allegedly pathognomonic of F alciparnm Malaria. Thedoctor and the medical technicians who observed the slides had nodifficulty in diagnosing the patient as having Falciparum Malaria. Inone instance, a trophozoite was found to be present in a reticulocyte.

In the heavy infection, gametocytes were found to be present. However,they did not have the typical crescent appearance of gametocytes whichappear in Falciparum Malaria. The gametocytes were mostly ovoid in shapewith dense staining chromatin material at one pole. However,

some of the gametocytes were dumb-bell shaped with the dense stainingchromatin material in the middle.

The doctor expressed his impression that it was easier to detect theparasites with pre-stained slides than it was on the conventional thinpreparation. The utilization of prestained slides for recognition andidentification of malarial parasites has the following advantages overthe existing method:

(1) Greater ease of recognition (2) Faster method for preparing slides(3) Slides can be prepared in the field without the need for laboratoryfacilities.

What is claimed is:

1. The method of identifying malarial parasites in human blood whichcomprises:

(a) applying a fresh sample of blood to a microscope slide having asurface coated with a dried mixture of 91 to '94 weight percentMethylene Blue NN and 6 to 9 weight percent Cresyl Violet Acetate;

(b) letting the slide with the blood sample so applied remain for a timesufficient to produce staining of the blood constituents;

(c) observing the blood constituents upon the slide.

2. The method of differentially staining malarial parasites in humanblood which comprises applying a sample of blood containing suchparasites to a surface bearing a dried mixture of 91 to 94 weightpercent Methylene Blue NN and 6 to 9 weight percent Cresyl VioletAcetate.

References Cited Sabin: Bull., The Johns Hopkins Hospital, vol. 34,1923, pp. 277-288.

Blecher, AJCP, vol. 19, 1949, pp. 895-896.

RONALD E. SERWIN, Primary Examiner U.S. Cl. X.R.

23-253 TP; l03.5 R; 252-408

